Frequency of the Mating-Type (MAT1) in Histoplasma capsulatum Isolates from Buenos Aires, Argentina.

Sex is genetically determined in Histoplasma capsulatum, governed by a sex-specific region in the genome called the mating-type locus (MAT1). We investigate the distribution of isolates of two H. capsulatum mating types in the clades circulating in Buenos Aires, Argentina. Forty-nine H. capsulatum isolates were obtained from the culture collection of the Mycology Center. The MAT1 locus was identified by PCR from the yeast suspension. The analysis of forty-eight isolates from clinical samples exhibited a ratio of 1.7 (MAT1-1:MAT1-2) and the only isolate from soil was MAT1-1. Forty-five H. capsulatum isolates belonged to the LAm B clade (H. capsulatum from Latin American group B clade) and showed a ratio of 1.8 (MAT1-1:MAT1-2). These results suggest an association between the mating types in isolates belonging to the LAm B clade. It remains to be defined whether a greater virulence should be attributed to the differences between the strains of the opposite mating type of the LAm B clade.

Dimorphism and Dissemination of Histoplasma capsulatum in the Upper Respiratory Tract after Intranasal Infection of Bats and Mice with Mycelial Propagules.

This article describes, for the first time, the role of the nasal mucosa (NM) as the initial site for the Histoplasma capsulatum mycelial-to-yeast transition. The results highlight that yeasts may arrive to the cervical lymph nodes (CLN) via phagocytes. Bats and mice were intranasally infected with H. capsulatum mycelial propagules and they were killed 10, 20, and 40 minutes and 1, 2, and 3 hours after infection. The NM and the CLN were monitored for fungal presence. Yeasts compatible with H. capsulatum were detected within the NM and the CLN dendritic cells (DCs) 2-3 hours postinfection, using immunohistochemistry. Histoplasma capsulatum was re-isolated by culturing at 28°C from the CLN of both mammalian hosts 2-3 hours postinfection. Reverse transcription-polymerase chain reaction assays were designed to identify fungal dimorphism, using mycelial-specific (MS8) and yeast-specific (YPS3) gene expression. This strategy supported fast fungal dimorphism in vivo, which began in the NM 1 hour postinfection (a time point when MS8 and YPS3 genes were expressed) and it was completed at 3 hours (a time point when only the YPS3 transcripts were detected) in both bats and mice. The presence of intracellular yeasts in the nasal-associated lymphoid tissue (NALT), in the NM nonassociated with the NALT, and within the interdigitating DCs of the CLN suggests early fungal dissemination via the lymph vessels.

Case Report: Hemophagocytic Lymphohistiocytosis Caused by Disseminated Histoplasmosis in a Venezuelan Patient with HIV and Epstein-Barr Virus Reactivation Who Traveled to Japan.

We describe a Venezuelan visitor to Japan who was diagnosed with hemophagocytic lymphohistiocytosis (HLH). The patient was also diagnosed with human immunodeficiency virus (HIV) and Epstein-Barr virus infection by the Western blot and polymerase chain reaction (PCR) tests, respectively. The cause of HLH was considered to be these two infections at first; however, the patient did not recover with antiretroviral/anti-herpes virus therapy. Thereafter, diagnosis of disseminated histoplasmosis was confirmed with an antigen detection test, culture, and PCR test of blood, urine, and bone marrow, and the patient improved gradually after the initiation of liposomal amphotericin B. This case highlights the importance of ruling out endemic mycosis as a cause of HLH even if other probable causes exist in patients from endemic areas.

Histoplasma capsulatum in planktonic and biofilm forms: in vitro susceptibility to amphotericin B, itraconazole and farnesol.

It is believed that most microbial infections are caused by pathogens organized in biofilms. Recently, it was shown that the dimorphic fungus Histoplasma capsulatum, estimated to be the most common cause of fungal respiratory diseases, is also able to form biofilm. Although the antifungal therapy commonly used is effective, refractory cases and recurrences have been reported. In the search for new compounds with antimicrobial activity, the sesquiterpene farnesol has gained prominence for its antifungal action. This study aimed to evaluate the in vitro susceptibility of H. capsulatum var. capsulatum to the antifungal agents itraconazole and amphotericin B, and farnesol alone and combined, as well as to determine the in vitro antifungal activity of these compounds against biofilms of this pathogen. The results show that farnesol has antifungal activity against H. capsulatum in the yeast and filamentous phases, with MIC values ranging from 0.0078 to 0.00312 µM. A synergistic effect (fractional inhibitory concentration index ≤0.5) between itraconazole and farnesol was found against 100 and 83.3 % of the isolates in yeast and mycelial forms, respectively, while synergism between amphotericin B and farnesol was only observed against 37.5 and 44.4 % of the isolates in yeast and filamentous forms, respectively. Afterwards, the antifungal drugs, itraconazole and amphotericin B, and farnesol alone, and the combination of itraconazole and farnesol, were tested against mature biofilms of H. capsulatum, through XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) metabolic assay, and the itraconazole and amphotericin B showed lower antibiofilm activity when compared to farnesol alone and farnesol combined with itraconazole. In conclusion, farnesol showed promising results as an antifungal agent against H. capsulatum and also showed adjuvant action, especially when combined with itraconazole, increasing the fungal susceptibility to this drug.

The importance of molecular analyses for understanding the genetic diversity of Histoplasma capsulatum: an overview.

Advances in the classification of the human pathogen Histoplasma capsulatum (H. capsulatum) (ascomycete) are sustained by the results of several genetic analyses that support the high diversity of this dimorphic fungus. The present mini-review highlights the great genetic plasticity of H. capsulatum. Important records with different molecular tools, mainly single- or multi-locus sequence analyses developed with this fungus, are discussed. Recent phylogenetic data with a multi-locus sequence analysis using 5 polymorphic loci support a new clade and/or phylogenetic species of H. capsulatum for the Americas, which was associated with fungal isolates obtained from the migratory bat Tadarida brasiliensis. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

Sexual variability in Histoplasma capsulatum and its possible distribution: what is going on?

Histoplasma capsulatum is a dimorphic fungal pathogen naturally found in the soil. Inhalation of conidia can result in pulmonary histoplasmosis and, in some cases, causes severe disseminated disease and death. This fungus is an ascomycete that has an anamorphic or asexual stage and a teleomorphic or sexual stage, known as Ajellomyces capsulatus, which results from (+) and (-) mating types. Sexual reproduction is regulated by a specialized genomic region known as the mating-type (MAT1) locus. The mating process in this heterothallic species is represented by isolates that contain only one of the two different MAT1 locus idiomorphs (MAT1-1 or MAT1-2) that have unrelated sequences encoding different transcription factors. In medically important dimorphic pathogens and in most ascomycete molds, one MAT locus idiomorph encodes a high-mobility-group (HMG) box-domain transcription factor, and the other idiomorph encodes an alpha-box domain transcription factor. There is scarce molecular information about H. capsulatum mating type although recombinant population structures have been reported that could occur in nature and this process has been documented in distinct models such as parasites and other fungi. In this review, we shall focus on published studies on H. capsulatum sexuality, and outline the distribution of the two H. capsulatum mating types in Latin America. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

Adhesion of Histoplasma capsulatum to pneumocytes and biofilm formation on an abiotic surface.

The pathogenic fungus, Histoplasma capsulatum, causes the respiratory and systemic disease 'histoplasmosis'. This disease is primarily acquired via inhalation of aerosolized microconidia or hyphal fragments of H. capsulatum. Evolution of this respiratory disease depends on the ability of H. capsulatum yeasts to survive and replicate within alveolar macrophages. It is known that adhesion to host cells is the first step in colonization and biofilm formation. Some microorganisms become attached to biological and non-biological surfaces due to the formation of biofilms. Based on the importance of biofilms and their persistence on host tissues and cell surfaces, the present study was designed to investigate biofilm formation by H. capsulatum yeasts, as well as their ability to adhere to pneumocyte cells. H. capsulatum biofilm assays were performed in vitro using two different clinical strains of the fungus and biofilms were characterized using scanning electron microscopy. The biofilms were measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay. The results showed that both the H. capsulatum strains tested were very efficient at adhering to host cells and forming biofilm. Therefore, this is a possible survival strategy adopted by this fungus.

Biological activity of the mite Sancassania sp. (Acari: Acaridae) from bat guano associated with the pathogenic fungus Histoplasma capsulatum.

Mites and the mammal pathogenic fungus Histoplasma capsulatum are the major components of bat guano microbiota. Interactions between mites and H. capsulatum were evaluated under laboratory conditions. Acarid mites, mainly Sancassania sp., were the most abundant microarthropod in the sampled guano of the Mexican bat Tadarida brasiliensis mexicana and, based on its morphology, Sancassania sp. was similar to the cosmopolitan species Sancassania sphaerogaster. The mycophagous and vectoring activities of this mite were tested for H. capsulatum and two other fungal species, Sporothrix schenckii (pathogenic) and Aspergillus sclerotiorum (non-pathogenic). S. ca. sphaerogaster was able to reproduce in H. capsulatum and S. schenckii colonies, multiplying in great numbers under controlled fungal mycelial-phase culture conditions. H. capsulatum colonies were completely destroyed after 14 days of in vitro interaction with mites. In contrast, S. ca. sphaerogaster did not reproduce in A. sclerotiorum cultures. S. ca. sphaerogaster was found vectoring H. capsulatum, but not the two other fungal species studied.

Adherence patterns of Histoplasma capsulatum yeasts to bat tissue sections.

The adherence of Histoplasma capsulatum yeasts to lung, spleen, liver, gut, and trachea cryosections of Artibeus hirsutus bats and inbred BALB/c mice (control) was studied after in vitro yeast-tissue incubations. Candida albicans yeasts were used as a well-known adherent fungal model in the mice host, and latex beads were used as a negative adherence control. Adhered yeast cells were identified by using crystal violet staining and the immunoperoxidase method with specific antibodies. H. capsulatum yeasts adhered to all tissues tested, mainly in the lung. Moreover, H. capsulatum yeasts adhered preferentially to white and red spleen pulp, in contrast to the dispersed distribution of C. albicans yeasts. H. capsulatum yeasts were mostly found on the sinusoidal face of hepatocytes. In general, the gut showed a higher number of adhered H. capsulatum yeasts than the trachea in both bats and mice. H. capsulatum and C. albicans yeasts developed high selectivity for the lamina propria of the gut. In addition, H. capsulatum yeasts interacted better with the lamina propria and adventitia of the trachea. The number of H. capsulatum yeast cells that adhered to each tissue section type was always greater than the corresponding number of C. albicans yeast cells, and latex beads never adhered to the tissue sections. Controls with anti-H. capsulatum and normal rabbit sera showed a significant blockage of H. capsulatum yeast adherence to lung tissue. This is the first study describing the patterns of H. capsulatum yeast adherence to different bat and mouse tissues.

Biological activity of the mite Sancassania sp. (Acari: Acaridae) from bat guano associated with the pathogenic fungus Histoplasma capsulatum

Mites and the mammal pathogenic fungus Histoplasma capsulatum are the major components of bat guano microbiota. Interactions between mites and H. capsulatum were evaluated under laboratory conditions. Acarid mites, mainly Sancassania sp., were the most abundant microarthropod in the sampled guano of the Mexican bat Tadarida brasiliensis mexicana and, based on its morphology, Sancassania sp. was similar to the cosmopolitan species Sancassania sphaerogaster. The mycophagous and vectoring activities of this mite were tested for H. capsulatum and two other fungal species, Sporothrix schenckii (pathogenic) and Aspergillus sclerotiorum (non-pathogenic). S. ca. sphaerogaster was able to reproduce in H. capsulatum and S. schenckii colonies, multiplying in great numbers under controlled fungal mycelial-phase culture conditions. H. capsulatum colonies were completely destroyed after 14 days of in vitro interaction with mites. In contrast, S. ca. sphaerogaster did not reproduce in A. sclerotiorum cultures. S. ca. sphaerogaster was found vectoring H. capsulatum, but not the two other fungal species studied.

Effect of anti-glycosphingolipid monoclonal antibodies in pathogenic fungal growth and differentiation. Characterization of monoclonal antibody MEST-3 directed to Manpalpha1-->3Manpalpha1-->2IPC.

BACKGROUND: Studies carried out during the 1990's demonstrated the presence of fungal glycoinositol phosphorylceramides (GIPCs) with unique structures, some of them showed reactivity with sera of patients with histoplasmosis, paracoccidioidomycosis or aspergillosis. It was also observed that fungal GIPCs were able to inhibit T lymphocyte proliferation "in vitro", and studies regarding the importance of these molecules to fungal survival showed that many species of fungi are vulnerable to inhibitors of sphingolipid biosynthesis. RESULTS: In this paper, we describe a detailed characterization of an IgG2a monoclonal antibody (mAb), termed MEST-3, directed to the Paracoccidioides brasiliensis glycolipid antigen Pb-2 (Manpalpha1-->3Manpalpha1-->2IPC). mAb MEST-3 also recognizes GIPCs bearing the same structure in other fungi. Studies performed on fungal cultures clearly showed the strong inhibitory activity of MEST-3 on differentiation and colony formation of Paracoccidioides brasiliensis, Histoplasma capsulatum and Sporothrix schenckii. Similar inhibitory results were observed when these fungi where incubated with a different mAb, which recognizes GIPCs bearing terminal residues of beta-D-galactofuranose linked to mannose (mAb MEST-1). On the other hand, mAb MEST-2 specifically directed to fungal glucosylceramide (GlcCer) was able to promote only a weak inhibition on fungal differentiation and colony formation. CONCLUSIONS: These results strongly suggest that mAbs directed to specific glycosphingolipids are able to interfere on fungal growth and differentiation. Thus, studies on surface distribution of GIPCs in yeast and mycelium forms of fungi may yield valuable information regarding the relevance of glycosphingolipids in processes of fungal growth, morphological transition and infectivity.

The interaction between Histoplasma capsulatum cell wall carbohydrates and host components: relevance in the immunomodulatory role of histoplasmosis.

Histoplasma capsulatum is an intracellular fungal pathogen that causes respiratory and systemic disease by proliferating within phagocytic cells. The binding of H. capsulatum to phagocytes may be mediated by the pathogen's cell wall carbohydrates, glucans, which consist of glucose homo and hetero-polymers and whose glycosydic linkage types differ between the yeast and mycelial phases. The alpha-1,3-glucan is considered relevant for H. capsulatum virulence, whereas the beta-1,3-glucan is antigenic and participates in the modulation of the host immune response. H. capsulatum cell wall components with lectin-like activity seem to interact with the host cell surface, while host membrane lectin-like receptors can recognize a particular fungal carbohydrate ligand. This review emphasizes the relevance of the main H. capsulatum and host carbohydrate-driven interactions that allow for binding and internalization of the fungal cell into phagocytes and its subsequent avoidance of intracellular elimination.

The interaction between Histoplasma capsulatum cell wall carbohydrates and host components: relevance in the immunomodulatory role of histoplasmosis

Histoplasma capsulatum is an intracellular fungal pathogen that causes respiratory and systemic disease by proliferating within phagocytic cells. The binding of H. capsulatum to phagocytes may be mediated by the pathogen's cell wall carbohydrates, glucans, which consist of glucose homo and hetero-polymers and whose glycosydic linkage types differ between the yeast and mycelial phases. The ±-1,3-glucan is considered relevant for H. capsulatum virulence, whereas the ²-1,3-glucan is antigenic and participates in the modulation of the host immune response. H. capsulatum cell wall components with lectin-like activity seem to interact with the host cell surface, while host membrane lectin-like receptors can recognize a particular fungal carbohydrate ligand. This review emphasizes the relevance of the main H. capsulatum and host carbohydrate-driven interactions that allow for binding and internalization of the fungal cell into phagocytes and its subsequent avoidance of intracellular elimination.

[Dimorphism and pathogenesis of Histoplasma capsulatum]. / Dimorfismo y patogenia de Histoplasma capsulatum.

Dimorphism and pathogenesis of Hisdistoplasma capsulatum is a dimorphic fungal pathogen with worldwide significance, which causes a broad spectrum of disease. In the saprophytic stage, it lives as a mycelial form consisting of hyphae bearing both macro and microconidia. Infection with H. capsulatum occurs by inhalation of microconidia (1-4 x 2-6 microm) or small mycelia fragments (5-8 microm) in the terminal bronchioles and alveoli of the lung. Inhaled conidia then convert into the yeast form that is responsible for the pathogenesis of histoplasmosis. As a soil fungus with no known requirements for interacting with a mammalian host as a necessary stage of its life cycle, the number of its strategies for successful pathogenesis is particularly remarkable. They include dimorphic mould-yeast transition, entry into host macrophages, subcellular localization, intracellular survival and proliferation during clinically unapparent infection with capacity for reactivation. H. capsulatum became the subject of increasing studies concurrently with the rising prevalence of human immunodeficiency. This paper presents an overall view of advances in the investigation of H. capsulatum dimorphic transition and pathogenesis.

Differential release of MIP-1alpha and eotaxin during infection of mice by Histoplasma capsulatum or inoculation of beta-glucan.

OBJECTIVE: In the present study, we evaluated the levels of MIP-1alpha and eotaxin and in vivo migration in the peritoneal cavity model, in mice inoculated with live yeast forms of Histoplasma capsulatum or the beta-glucan cell wall component of this fungus, and the influence of a leukotriene biosynthesis inhibitor, MK886, on the release of these chemokines in relation to cell recruitment. MATERIALS: Female outbred Swiss mice (N = 4-5 per group, 3-4 wk, were used. Mice were injected i.p. with 1 ml of the 6 x 10(5) live yeast form of the fungus or with 10 microg of beta-glucan from the cell wall fraction, and treated daily with MK886 (1 mg kg(-1), p.o.) or vehicle. RESULTS: The fungus induced rapid generation of high levels of MIP-1alpha, which remained elevated from 4-48 h whereas very little eotaxin was detected at any time point (Fig. 1A and B). In contrast, the beta-glucan induced a little MIP-1alpha but considerably higher concentrations of eotaxin within the first four hours; however, the level of neither chemokine was sustained (Fig. 2A and B). Treatment of animals with MK886 was effective in reducing the numbers of neutrophils, eosinophils and, to a lesser degree, mononuclear cells accumulating in the peritoneal cavity in response to both the live fungus (Fig. 1C-E) and the cell wall beta-glucan (Fig. 2C-E). CONCLUSIONS: The results suggest that chemokines and leukotrienes may play key roles in the inflammatory cell influx to H. capsulatum infection or to the inoculation of the beta-glucan cell wall component of this fungus

[Acute pulmonary histoplasmosis in a Spanish traveller to Nicaragua: an imported disease case]. / Histoplasmosis pulmonar aguda en un viajero español a Nicaragua: ejemplo de enfermedad importada.

Pulmonary histoplasmosis is a rare disease in Spain. Moreover, it is difficult to diagnose due to unspecific clinical and radiological symptoms. The isolation of the fungus is essential for a proper diagnosis. Nevertheless, it is very difficult to identify the fungus itself in respiratory stains and we usually need invasive techniques. For all these reasons and taking into account the increase in journeys and immigration, we believe that it is probably underdiagnosed in our country. We report a case of acute pulmonary histoplasmosis in a Spanish traveller and emphasize the importance of the anamnesis and the value of the microbiologist's experience to obtain the diagnosis.

Histoplasma capsulatum inhibits apoptosis and Mac-1 expression in leucocytes.

Histoplasma capsulatum is a fungus found intracellularly in neutrophils and peripheral blood mononuclear cells (PBMCs), suggesting that it is capable of evading damage and survives inside these cells. In this study, we report that neutrophils from H. capsulatum-infected mice, and human neutrophils and mononuclear cells exposed to H. capsulatum presented less apoptosis than those from noninfected animals or cells exposed to medium only. Moreover, cells harvested from infected animals are resistant to apoptosis induced by dexamethasone - a proapoptotic stimulant. We also show that neutrophils harvested from infected mice and PBMCs from humans exposed to the fungus had a greatly decreased Mac-1 expression. We conclude that H. capsulatum induces an antiapoptotic state on leucocytes, which correlates with decreased cell-surface Mac-1 expression. These facts may represent an escape mechanism for the fungus by delaying cell death and allowing the fungus to survive inside leucocytes.

Pathogenesis II: fungal responses to host responses: interaction of host cells with fungi.

Most of our knowledge concerning the virulence determinants of pathogenic fungi comes from the infected host, mainly from animal models and more recently from in vitro studies with cell cultures. The fungi usually present intra- and/or extracellular host-parasite interfaces, with the parasitism phenomenon dependent on complementary surface molecules. Among living organisms, this has been characterized as a cohabitation event, where the fungus is able to recognize specific host tissues acting as an attractant, creating stable conditions for its survival. Several fungi pathogenic for humans and animals have evolved special strategies to deliver elements to their cellular targets that may be relevant to their pathogenicity. Most of these pathogens express surface factors that mediate binding to host cells either directly or indirectly, in the latter case binding to host adhesion components such as extracellular matrix (ECM) proteins, which act as 'interlinking' molecules. The entry of the pathogen into the host cell is initiated by fungal adherence to the cell surface, which generates an uptake signal that may induce its cytoplasmic internalization. Once this is accomplished, some fungi are able to alter the host cytoskeletal architecture, as manifested by a rearrangement of microtubule and microfilament proteins, and this can also induce epithelial host cells to become apoptotic. It is possible that fungal pathogens induce modulation of different host cell pathways in order to evade host defences and to foster their own proliferation. For a number of pathogens, the ability to bind ECM glycoproteins, the capability of internalization and the induction of apoptosis are considered important factors in virulence. Furthermore, specific recognition between fungal parasites and their host cell targets may be mediated by the interaction of carbohydrate-binding proteins, e.g., lectins on the surface of one type of cell, probably a parasite, that combine with complementary sugars on the surface of host-cell. These interactions supply precise models to study putative adhesins and receptor-containing molecules in the context of the fungus-host interface. The recognition of the host molecules by fungi such as Aspergillus fumigatus, Paracoccidioides brasiliensis and Histoplasma capsulatum, and their molecular mechanisms of adhesion and invasion, are reviewed in this paper.

[Strategies of Histoplasma capsulatum for evading the cytocidal mechanisms of phagocytes]. / Estrategias del Histoplasma capsulatum para evadir los mecanismos citocidas de los fagocitos.

Although proliferation of Histoplasma capsulatum within the macrophage is restricted by cell mediated immunity and, that there is no apparent deficiency in the fungicidal power of the macrophage, H. capsulatum can prosper in this intracellular microenvironment in some circumstances, which provides special nutritional advantages to intracellular growth for H. capsulatum, and access to other organs via the bloodstream and lymphatics. Nevertheless the environment within professional phagocytes is complex and generally hostile to microorganisms. Intracellular pathogens must avoid or overcome a series of obstacles in order to prevent their destruction. We present the strategies used by H. capsulatum to escape the host cell aggressiveness from the moment it reaches the cell surface up to its survival inside the phagocyte. Our aim is to discuss the advances on the escape mechanisms used by H. capsulatum to survive in the intracellular environment.

A medium for inducing conversion of Histoplasma capsulatum var. capsulatum into its yeast-like form

Histoplasma capsulatum var. capsulatum is a dimorphic fungus that, under special conditions, converts from its more common mycelial form to a yeast-like form. Achieving this conversion, however, has been problematical for researchers. The present study tested conversion rates in ten Histoplasma capsulatum var. capsulatum strains using seven culture media, four of wich were conventional and three novel. One of our novel media, MLGema, induced complete conversion, of two strains within five days of incubation at 35 degrees centigrades, and of all strains that eventually converted by the time of the second subculturing transfer, under defined experimental conditions. MLGema is also inexpensive and easy to produce