Histoplasmosis Outbreaks in Brazil: Lessons to Learn About Preventing Exposure.

Histoplasmosis is considered the most common invasive opportunistic fungal disease in the Americas, with outbreaks and micro-epidemics reported for over 80 years. In Brazil, this disease has been described since 1946, reaching a remarkable incidence in the population, especially during the HIV-AIDS pandemic. In this study, published and unpublished outbreaks and micro-epidemics of histoplasmosis in Brazil were revisited by accessing different database sources and evaluating epidemiological and clinical features. We have found reports spanning 1946-2017, across 10 Brazilian states and with involvement of 370 humans and 2 dogs, and 13 disseminated cases and 3 deaths were reported. Rio de Janeiro had the largest number of outbreaks (n = 20/40; 50%) reported in this study. The majority of outbreaks and micro-epidemics was reported in caves (n = 21/40; 52.5%), followed by reports in abandoned/deactivated sites (n = 6/40; 15%), mines (n = 5/40; 12.5%), chicken coops (n = 4/40; 10%). Histoplasmosis is a serious health issue in Brazil considering the attractive and growing market of ecotourism throughout more than 7000 caves, and all levels of poultry farming activity are important to raise awareness about how dangerous this neglected disease can be and establish ways to decrease exposure to contaminated environmental sources through adequate preventive measures.

Modification of the Francis Medium for the Conversion of Histoplasma capsulatum to the Yeast-Like Growth Phase.

We propose a modification of Francis agar used for identification of the causative agent of histoplasmosis by in vitro conversion of the mycelial culture to the yeast-like growth phase. For improving of the growth characteristics of the medium, we used FT-agar with glucose-vitamin additives developed for culturing of the tularemia causative agent. The modified Francis medium is characterized by significantly higher growth properties and allowed 10-fold increasing the number of CFU of yeast-like cells of both American and African histoplasmosis causative agent.

C-type lectin receptors differentially induce th17 cells and vaccine immunity to the endemic mycosis of North America.

Vaccine immunity to the endemic mycoses of North America requires Th17 cells, but the pattern recognition receptors and signaling pathways that drive these protective responses have not been defined. We show that C-type lectin receptors exert divergent contributions to the development of antifungal Th17 cells and vaccine resistance against Blastomyces dermatitidis, Histoplasma capsulatum, and Coccidioides posadasii. Acquired immunity to B. dermatitidis requires Dectin-2, whereas vaccination against H. capsulatum and C. posadasii infection depends on innate sensing by Dectin-1 and Dectin-2, but not Mincle. Tracking Ag-specific T cells in vivo established that the Card9 signaling pathway acts indispensably and exclusively on differentiation of Th17 cells, while leaving intact their activation, proliferation, survival, and migration. Whereas Card9 signaling is essential, C-type lectin receptors offer distinct and divergent contributions to vaccine immunity against these endemic fungal pathogens. Our work provides new insight into innate immune mechanisms that drive vaccine immunity and Th17 cells.

Immunization with apoptotic phagocytes containing Histoplasma capsulatum activates functional CD8(+) T cells to protect against histoplasmosis.

We have previously revealed the protective role of CD8(+) T cells in host defense against Histoplasma capsulatum in animals with CD4(+) T cell deficiency and demonstrated that sensitized CD8(+) T cells are restimulated in vitro by dendritic cells that have ingested apoptotic macrophage-associated Histoplasma antigen. Here we show that immunization with apoptotic phagocytes containing heat-killed Histoplasma efficiently activated functional CD8(+) T cells whose contribution was equal to that of CD4(+) T cells in protection against Histoplasma challenge. Inhibition of macrophage apoptosis due to inducible nitric oxide synthase (iNOS) deficiency or by caspase inhibitor treatment dampened the CD8(+) T cell but not the CD4(+) T cell response to pulmonary Histoplasma infection. In mice subcutaneously immunized with viable Histoplasma yeasts whose CD8(+) T cells are protective against Histoplasma challenge, there was heavy granulocyte and macrophage infiltration and the infiltrating cells became apoptotic. In mice subcutaneously immunized with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled apoptotic macrophages containing heat-killed Histoplasma, the CFSE-labeled macrophage material was found to localize within dendritic cells in the draining lymph node. Moreover, depleting dendritic cells in immunized CD11c-DTR mice significantly reduced CD8(+) T cell activation. Taken together, our results revealed that phagocyte apoptosis in the Histoplasma-infected host is associated with CD8(+) T cell activation and that immunization with apoptotic phagocytes containing heat-killed Histoplasma efficiently evokes a protective CD8(+) T cell response. These results suggest that employing apoptotic phagocytes as antigen donor cells is a viable approach for the development of efficacious vaccines to elicit strong CD8(+) T cell as well as CD4(+) T cell responses to Histoplasma infection.

Pulmonary histoplasmosis.

Pulmonary histoplasmosis is an important cause of morbidity in the United States. Several outbreaks of acute pulmonary histoplasmosis have been linked to potentially preventable environmental exposures. Progressive disseminated histoplasmosis, which is seen frequently in the growing population of immunocompromised hosts, often presents with prominent pulmonary manifestations and is more commonly encountered in hospitalized patients than acute, subacute, or chronic pulmonary histoplasmosis. A battery of diagnostic studies including serology, antigen, cytology/histopathology, and culture should be obtained in suspected cases of histoplasmosis. The yield of antigenuria detection is highest when the multiple body fluids are tested; the level of antigenuria correlates with severity of disease. Amphotericin B is the treatment of choice for severe pulmonary or disseminated histoplasmosis, and itraconazole is effective for mild to moderately severe infection. Posaconazole exhibits promise as a salvage agent. Antifungal prophylaxis is not routinely recommended for at-risk populations. Measures to minimize environmental contamination may reduce the risk of epidemic-type acute pulmonary histoplasmosis related to high-risk exposures.

The PD-1/PD-L costimulatory pathway critically affects host resistance to the pathogenic fungus Histoplasma capsulatum.

The PD-1 costimulatory receptor inhibits T cell receptor signaling upon interacting with its ligands PD-L1 and PD-L2. The PD-1/PD-L pathway is critical in maintaining self-tolerance. In this study, we examined the role of PD-1 in a mouse model of acute infection with Histoplasma capsulatum, a major human pathogenic fungus. In a lethal model of histoplasmosis, all PD-1-deficient mice survived infection, whereas the wild-type mice died with disseminated disease. PD-L expression on macrophages and splenocytes was up-regulated during infection, and macrophages from infected mice inhibited in vitro T cell activation. Of interest, antibody blocking of PD-1 significantly increased survival of lethally infected wild-type mice. Thus, our studies extend the role of the PD-1/PD-L pathway in regulating antimicrobial immunity to fungal pathogens. The results show that the PD-1/PD-L pathway has a key role in the regulation of antifungal immunity, and suggest that manipulation of this pathway represents a strategy of immunotherapy for histoplasmosis.

Recognition, diagnosis, and treatment of histoplasmosis complicating tumor necrosis factor blocker therapy.

Life-threatening histoplasmosis is one of the most common opportunistic infections in patients receiving tumor necrosis factor (TNF) blockers. Delays in considering the diagnosis may lead to increased morbidity and mortality. Most affected patients present with pneumonitis, usually accompanied by additional signs of progressive dissemination, or with signs of progressive dissemination alone. The diagnosis often can be promptly established using antigen detection or direct examination of bronchoalveolar lavage specimens. If histoplasmosis is diagnosed promptly, antifungal therapy is highly effective. After a favorable clinical response, the safety of both discontinuation of antifungal therapy and the resumption of TNF blocker remains undetermined. The management of the immune reconstitution inflammatory syndrome that may follow discontinuation of TNF blockers also requires investigation. Prescribers should become aware of the recognition, diagnosis, and treatment of histoplasmosis and educate recipients about decreasing their risk of exposure and both recognizing and reporting signs of early infection.

CD40 ligand is not essential for induction of type 1 cytokine responses or protective immunity after primary or secondary infection with histoplasma capsulatum.

The induction of type 1 immune responses (interleukin [IL]-12, interferon [IFN]-gamma) has been shown to be important in mediating protection against many intracellular infections including Histoplasma capsulatum. Costimulatory molecules such as CD40 ligand (CD40L) have been shown to be a central regulator of type 1 responses in vivo. To study the role of CD40L in mediating protection against infection with H. capsulatum, CD40L-deficient (CD40L-/-) and CD40L+/+ mice were infected with H. capsulatum and assessed for various parameters. After a lethal challenge of H. capsulatum, CD40L-/- mice were not substantially different from CD40L+/+ mice in terms of mortality, fungal burden, or production of IFN-gamma, IL-12, nitric oxide, or tumor necrosis factor alpha. Moreover, CD40L-/- mice treated with anti-IFN-gamma or anti-IL-12 at the time of infection had accelerated mortality, providing further evidence that IL-12 and IFN-gamma are produced in vivo in the absence of CD40L. In addition, CD40L-/- mice infected with a sublethal dose of H. capsulatum survived infection, whereas all mice infected with the same dose and treated with anti-IFN-gamma had accelerated mortality, demonstrating that IFN-gamma but not CD40L was essential for primary immunity to H. capsulatum infection. Interestingly, depletion of either CD4+ or CD8+ T cells resulted in accelerated mortality in CD40L-/- mice, suggesting a critical role for these cells in response to infection. Finally, CD40L-/- mice initially infected with a sublethal dose of H. capsulatum were protected from secondary infection with a lethal dose of H. capsulatum, demonstrating that CD40L is not required for the maintenance of memory immunity.

Vaccine immunity to pathogenic fungi overcomes the requirement for CD4 help in exogenous antigen presentation to CD8+ T cells: implications for vaccine development in immune-deficient hosts.

Systemic fungal infections with primary and opportunistic pathogens have become increasingly common and represent a growing health menace in patients with AIDS and other immune deficiencies. T lymphocyte immunity, in particular the CD4+ Th 1 cells, is considered the main defense against these pathogens, and their absence is associated with increased susceptibility. It would seem illogical then to propose vaccinating these vulnerable patients against fungal infections. We report here that CD4+ T cells are dispensable for vaccine-induced resistance against experimental fungal pulmonary infections with two agents, Blastomyces dermatitidis an extracellular pathogen, and Histoplasma capsulatum a facultative intracellular pathogen. In the absence of T helper cells, exogenous fungal antigens activated memory CD8+ cells in a major histocompatibility complex class I-restricted manner and CD8+ T cell-derived cytokines tumor necrosis factor alpha, interferon gamma, and granulocyte/macrophage colony-stimulating factor-mediated durable vaccine immunity. CD8+ T cells could also rely on alternate mechanisms for robust vaccine immunity, in the absence of some of these factors. Our results demonstrate an unexpected plasticity of immunity in compromised hosts at both the cellular and molecular level and point to the feasibility of developing vaccines against invasive fungal infections in patients with severe immune deficiencies, including those with few or no CD4+ T cells.

Leukotrienes are potent adjuvant during fungal infection: effects on memory T cells.

Leukotrienes (LTs) are potent lipid mediators involved in the control of host defense. LTB(4) induces leukocyte accumulation, enhances phagocytosis and bacterial clearance, and increases NO synthesis. LTB(4) is also important in early effector T cell recruitment that is mediated by LTB(4) receptor 1, the high-affinity receptor for LTB(4). The aims of this study were to evaluate whether LTs are involved in the secondary immune response to vaccination in a murine model of Histoplasma capsulatum infection. Our results demonstrate that protection of wild-type mice immunized with cell-free Ags from H. capsulatum against histoplasmosis was associated with increased LTB(4) and IFN-gamma production as well as recruitment of memory T cells into the lungs. In contrast, cell-free Ag-immunized mice lacking 5-lipoxygenase(-/-), a critical enzyme involved in LT synthesis, displayed a marked decrease on recruitment of memory T cells to the lungs associated with increased synthesis of TGF-beta as well as IL-10. Strikingly, these effects were associated with increased mortality to 5-lipoxygenase(-/-)-infected mice. These data establish an important immunomodulatory role of LTs, in both the primary and secondary immune responses to histoplasmosis.

HIV-associated histoplasmosis in a nonendemic area of the United States during the HAART era: role of migration from endemic areas and lack of antiretroviral therapy.

Histoplasmosis is known to be an AIDS-associated infection, with scattered areas of endemicity throughout the world. Although the Atlanta, GA, metropolitan area is not a highly endemic area, a significant number of cases have been noted at our institution in recent years. Cases of histoplasmosis over a 4-year period were reviewed. All 27 patients (100%) were HIV infected. Thirty percent of patients with histoplasmosis were from Latin American countries. Patients from Latin America were younger than patients from the United States, tended to be more likely to have proven disease, and were exclusively male. Patients with proven disease had significantly higher urine histoplasma antigen levels, lower platelets counts, and lower neutrophil counts than patients with probable disease. The majority of patients survived after treatment with antifungals and initiation of antiretroviral therapy. Histoplasmosis is thus an important consideration in the workup of patients with advanced HIV in nonendemic areas of the United States.

Histoplasmosis in solid organ transplant recipients: 10 years of experience at a large transplant center in an endemic area.

BACKGROUND: Many clinical scenarios have been encountered by patients who developed histoplasmosis after receiving a solid organ transplant at a large transplant center in an endemic area. METHODS: Cases of posttransplantation histoplasmosis were identified by use of multiple methods, including reviews of microbiology test results, transplant databases, and billing codes. Data were obtained retrospectively. Descriptive statistics were used. RESULTS: During the 1997-2007 study period, 3436 patients received a solid organ transplant, and 38 patients were identified as having posttransplantation histoplasmosis. Of these 38 patients, 9 were excluded from our study because the diagnosis was solely clinical. Of the remaining 29 patients, 14 had posttransplantation histoplasmosis (incidence, 1 case per 1000 person-years); 14 showed histologic evidence of histoplasmosis in the recipient or donor tissue, which was encountered unexpectedly at the time of transplantation; and 1 had histoplasmosis before receiving the transplant. Of the 14 patients who developed histoplasmosis after transplantation, 5 were heart transplant recipients, 3 were lung transplant recipients, 3 were kidney transplant recipients, 1 was a liver transplant recipient, 1 was a pancreas transplant recipient, and 1 was a kidney-pancreas transplant recipient. The median time from transplantation to diagnosis was 17 months (interquartile range, 8.1-46 months), and the median time from onset of symptoms to diagnosis 3 weeks (interquartile range, 1.9-6.5 weeks). All recipients had disseminated disease. The most common treatment was amphotericin B and itraconazole. All were cured, or still on treatment, but symptom-free. Of the 14 patients who had an explanted organ or donor tissue that showed histologic evidence of histoplasmosis, 13 (93%) were lung transplant recipients, and 1 (7%) was a liver transplant recipient. None of these patients developed active histoplasmosis, but all received prophylactic treatment. Finally, 1 patient had histoplasmosis before transplantation; he was treated with itraconazole 3 months before and after transplantation, and he did well. CONCLUSIONS: In conclusion, posttransplantation histoplasmosis is rare (1 case per 1000 transplant-person-years; 95% confidence interval, 0.6-1.7), even in endemic areas. Prognosis is good but requires protracted therapy. Patients with latent infection did not develop posttransplantation histoplasmosis when prophylaxis was used.

Vaccination with an alkaline extract of Histoplasma capsulatum packaged in glucan particles confers protective immunity in mice.

Infection with the dimorphic fungus, Histoplasma capsulatum, occurs world-wide, but North and South America are regions of high endemicity. Interventions to mitigate exposure and consequent disease are limited to remediating a habitat harboring the fungus. The development of a vaccine to prevent infection or lessen its severity is an important advance in disease prevention. Accordingly, we prepared an alkaline extract from the yeast phase of Histoplasma and encased it in glucan particles that act as an adjuvant and delivery vehicle. Immunization of C57BL/6 mice with this encapsulated extract decreased the number of CFUs in lungs and spleens at days 7 and 14 following intranasal infection. Moreover, this vaccine conferred protection against a lethal challenge with the fungus. Cytokine assessment in lungs at a time when the CFUs were similar between controls and vaccinated groups revealed increased quantities of interferon-γ and interleukin-17 in vaccine recipients. This finding was supported by increased generation of both Th1 and Th17 cells in lungs and draining lymph nodes of vaccinated mice compared to controls. Neutralization of interferon-γ or interleukin-17 blunted the effectiveness of vaccination. To identify the proteins comprising this extract, liquid chromatography tandem mass spectrometry was performed. Thus, an H. capsulatum alkaline extract packaged in glucan particles confers protection in an interferon-γ and interleukin-17-dependent manner. Discovery of a single protein or a few proteins in this admixture that mediate protective immunity would represent significant progress in efforts to prevent histoplasmosis.

Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum.

A protective role for antibodies has not previously been described for host defense against the pathogenic fungus Histoplasma capsulatum (Hc). Mouse mAb's were generated from mice immunized with Hc yeast that binds the cell surface of Hc. Administration of mAb's before Hc infection reduced fungal burden, decreased pulmonary inflammation, and prolonged survival in a murine infection model. Protection mediated by mAb's was associated with enhanced levels of IL-4, IL-6, and IFN-gamma in the lungs of infected mice. The mAb's increased phagocytosis of yeast by J774.16 cells through a CR3-dependent process. Ingestion of mAb-opsonized Hc by J774.16 macrophage-like cells was associated with yeast cell growth inhibition and killing. The mAb's bound to a 17-kDa antigen expressed on the surface of Hc. The antigen was identified as a histone H2B-like protein. This study establishes that mAb's to a cell surface protein of Hc alter the intracellular fate of the fungus and mediate protection in a murine model of lethal histoplasmosis, and it suggests a new candidate antigen for vaccine development.

[Efficacy of the treatment and secondary antifungal prophylaxis in AIDS-related histoplasmosis. Experience at the Francisco J. Muñiz Infectious Diseases Hospital in Buenos Aires]. / Eficacia del tratamiento y de la profilaxis antifúngica secundaria en la histoplasmosis asociada al sida. Experiencia del Hospital de Infecciosas Francisco J. Muñiz, de la ciudad de Buenos Aires.

BACKGROUND: Classic histoplasmosis is a systemic endemic mycosis due to Histoplasma capsulatum var. capsulatum. A significant reduction in the morbidity and mortality of AIDS-related histoplasmosis has been observed since the introduction of highly active antiretroviral therapy (HAART) and secondary antifungal prophylaxis. AIMS: The aim of this study was to determine the current state of prognosis and treatment response of HIV-positive patients with histoplasmosis in the Francisco J. Muñiz Infectious Diseases Hospital in Buenos Aires City. METHODS: A retrospective study was conducted using the demographic, clinical, immunological and treatment data of 80 patients suffering from AIDS-related histoplasmosis. RESULTS: Of the 80 cases studied 65 were male, the median age was 36 years, with 73.7% of the patients being drug addicts, 82.5% of the patients was not receiving HAART at diagnosis, and 58.7% of the cases had less than 50 CD4+ cells/µl at the beginning of the treatment. The initial phase of treatment consisted of intravenous amphotericin B and/or oral itraconazole for 3 months, with 78.7% of the cases showing a good clinical response. Only 26/63 patients who were discharged from hospital continued with the follow-up of the HAART, secondary prophylaxis with itraconazole or amphotericin B. Secondary prophylaxis was stopped after more than one year of HAART if the patients were asymptomatic, had two CD4+ cell counts greater than 150cells/µl, and undetectable viral loads. No relapses were observed during a two-year follow up after prophylaxis was stopped. CONCLUSIONS: The treatment of histoplasmosis in HIV-positive patients was effective in 78.8% of the cases. The combination of HAART and secondary antifungal prophylaxis is safe, well tolerated, and effective. The low adherence of patients to HAART and the lack of laboratory kits for rapid histoplasmosis diagnosis should be addressed in the future. The usefulness of primary antifungal prophylaxis for cryptococcosis and histoplasmosis HIV-positive patients should be studied.

Protective antibodies and endemic dimorphic fungi.

The host response to infection is the outcome of a complex interaction between a microbe and a host's innate and adaptive immune system. In this context, the role of antibody in the endemic mycoses is relatively poorly understood. Recently, a monoclonal antibody to a cell surface protein has been shown to be protective in a murine histoplasmosis model. The findings with Histoplasma capsulatum may provide a paradigm for antibody protection against endemic fungi. This paper reviews the recent data on protective antibody in histoplasmosis and previous data supporting a role for antibody in protective responses in other dimorphic fungi.

Preventing reactivation of ocular histoplasmosis: guidance for patients at risk.

BACKGROUND: Ocular histoplasmosis syndrome (OHS), a significant cause of vision loss in young and middle-aged adults, is associated with the fungus Histoplasma capsulatum (Hc). There is considerable evidence that recurrent reactivation of perimacular ocular histoplasmosis lesions is an important cause of disease progression and that vision loss is at least, in part, a consequence of host sensitivity to fungal antigen. METHODS: The etiology and pathogenesis of OHS is reviewed and specific recommendations are made for patients with OHS that may decrease the risk of reactivation of ocular histoplasmosis lesions and slow disease progression. CONCLUSION: Patients with perimacular chorioretinal scars secondary to OHS should be informed by the clinician that they are at risk for vision loss; they should be told the symptoms of choroidal neovascularization and how to self-monitor their vision with an Amsler grid. We recommend they also be instructed on how to decrease their risk of reinfection by Hc. Aggressive treatment of dermatomycoses, onychomycosis, vaginal candidiasis, and other chronic fungal infections may decrease the risk of reactivation of ocular lesions. Patients with OHS who are considering LASIK surgery should be informed that the procedure may trigger choroidal neovascularization.

Efficacy of cell-free antigens in evaluating cell immunity and inducing protection in a murine model of histoplasmosis.

Histoplasma capsulatum is a dimorphic pathogenic fungus that causes a wide spectrum of disease when mycelial fragments are inhaled. Resistance to H. capsulatum is dependent on cellular immunity mediated by T cells and macrophages. Here we standardized the production of extracts containing cell-free antigens (CFAgs) and observed their efficacy in evaluating cellular immunity during murine histoplasmosis. CFAgs induced a more potent delayed-type hypersensitivity (DTH) response in H. capsulatum-infected mice than did histoplasmin-a classical antigen. This DTH response to CFAgs is able to determine the immune status of infected mice and to predict their death. Moreover, CFAgs stimulated spleen cells from immune mice to produce higher amounts of gamma interferon (IFN-gamma) in vitro. Finally, immunization with CFAgs protected against a lethal inoculum of H. capsulatum. These results demonstrate that CFAgs may be useful for the evaluation of cellular immune response and as a potential source for the development of a vaccine against histoplasmosis.

Safety of discontinuation of maintenance therapy for disseminated histoplasmosis after immunologic response to antiretroviral therapy.

We performed a prospective observational study to assess the safety of stopping maintenance therapy for disseminated histoplasmosis among human immunodeficiency virus infected patients after response to antiretroviral therapy. All subjects received at least 12 months of antifungal therapy and 6 months of antiretroviral therapy before entry. Negative results of fungal blood cultures, urine and serum Histoplasma antigen level of <4.1 units, and CD4+ T cell count of >150 cells/mm3 were required for eligibility. Thirty-two subjects were enrolled; the median CD4+ T cell count at study entry was 289 cells/mm3. No relapses of histoplasmosis occurred after a median duration of follow-up of 24 months. This corresponded to an observed relapse rate of 0 cases per 65 person-years. The median CD4+ T cell count at final study visit was 338 cells/mm3. Discontinuation of antifungal maintenance therapy appears to be safe for patients with acquired immunodeficiency syndrome with previously treated disseminated histoplasmosis and sustained immunologic improvement in response to antiretroviral therapy.